1. Field of the Invention
The present invention relates to a method for obtaining a bacterium having an optimized level of gene expression, which is useful for amino acid or nucleic acid production, and a method for producing an L-amino acid or nucleic acid using the bacterium.
2. Brief Description of the Related Art
Traditionally, enhancing the activity of gene products involved in biosynthetic pathways of L-amino acids or nucleic acids was a common method used to increase L-amino acid or nucleic acid production. This was often accomplished by creating mutants resistant to target compounds or analogs, enhancing the expression of the biosynthetic genes, eliminating sensitivity of biosynthetic enzymes to feedback inhibition by products and intermediates of the biosynthesis, and creating bacterial strains deficient in genes which encode enzymes using the precursors of the target compound for other pathways, or creating bacterial strains deficient in genes which are responsible for the degradation of the target compound.
These manipulations usually lead to the creation of strains which cannot grow, or grow only at a significantly reduced rate, or require additional nutrients such as amino acids. For example, enhancement of expression of some genes may become excessive and could lead to significant inhibition of bacterial growth and, as a result, lower the ability of the bacterium to produce the target compound.
It is known that microorganisms belonging to the genus Escherichia which are deficient in or possess a decreased level of α-ketoglutarate dehydrogenase, have the ability to produce L-glutamic acid (U.S. Pat. Nos. 5,573,945 and 5,908,768). But these mutants cannot grow or are only able to grow at significantly reduced rates in glucose minimal media under aerobic conditions. Addition of succinic acid or lysine supplemented with methionine is necessary to restore growth. Therefore, the selection of an optimal expression level for α-ketoglutarate dehydrogenase is necessary for the bacterium to acquire the abilities to produce glutamic acid and to grow in a medium containing no additional supplements, such as succinic acid, lysine, or methionine.
On the other hand, it is known that genes encoding succinate dehydrogenase (sdhCDAB genes) as well as genes encoding α-ketoglutarate dehydrogenase (sucAB genes) and succinyl-CoA synthase (sucCD genes) form a cluster containing two promoters in the chromosome of E. coli: Psdh-sdhCDAB-Psuc-sucAB-sucCD (Park, S. J., Chao, G., and Gunsalus, R. P., J. Bact. 179, 4138-4142, 1997; Cunningham, L., and Guest, G. R., Microbiology, 144, 2113-2123, 1998). The main promoter for this operon structure is the regulatory region located upstream of the sdhC gene, Psdh. The weaker Psuc-promoter recognized by E. coli RNA polymerase in the complex with σ38 provides an additional low constitutive level of sucABCD gene expression. The activity of the later promoter has been relatively unaffected by the growth substrate. In addition, the effects of anaerobiosis and arcA or fnr mutations are relatively small. ArcA protein is a negative response regulator of genes in aerobic pathways, and Fnr (fumarate and nitrate reduction) protein is involved in transcriptional regulation of aerobic, anaerobic respiration and osmotic balance. Among potential regulators tested, IHF protein (integration host factor—product of himA gene) might play the major role in repressing Psuc activity. ArcA and Fnr proteins interact with Psdh promoter and IHF protein interacts with weaker Psuc promoter.
Obtaining a library of synthetic promoters for Lactococcus lactis with different strengths has been described (Jensen P. R., and Hammer K., Appl. Environ. Microbiol., 1998, 64, No. 1. 82-87 Biotechnol. Bioeng., 1998, 58, 2-3, 191-5). The library consists of 38 oligonucleotides having randomized spacers between the consensus sequences in the positions from −35 to −15. To evaluate the strength of the resulting promoters, oligonucleotides from the library were cloned into the expression vector pAK80, which contains β-galactosidase. It was shown that the majority of artificial promoters were very weak (below 500) and only three of them had a strength of about 2000 relative units. But no practical application for this library of synthetic promoters was disclosed.
A method for producing coryneform bacteria having an improved amino acid- or nucleic acid-productivity by introducing a mutation into a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes is disclosed in the European Patent Application EP1033407A1. Up to 8 different variants of mutant promoters were used for each of the following genes: glutamate dehydrogenase (gdh) gene, citrate synthase (gltA) gene, isocitrate dehydrogenase(icd) gene, pyruvate dehydrogenase (pdhA) gene, and argininosuccinate synthase (argG) gene. The disadvantage of the technique described in European application EP1033407A1 is that each of described mutants was prepared separately, that is one by one. Also, increasing L-amino acid production in all cases disclosed in this patent application was achieved by increasing the activity of certain enzymes using a limited amount of different promoters for the gene encoding the enzyme. This approach is within the mainstream of work focused on preparing L-amino acid or nucleic-acid producing bacteria, and does not pertain to optimization or fine-tuning of promoter activity of genes essential for production of L-amino acids or nucleic acids.
There have been no reports describing the optimization of gene expression for metabolite production by fermentation of a bacterium which has been modified to have an optimized level of target gene expression.